Receptor-associated protein (RAP), also known as alpha-2-macroglobulin/low density lipoprotein receptor-related protein-associated protein 1 (Uniprot accession P30533, Pfam accession numbers PF06400 and PF06401), is a unique 39 kD protein that binds to almost all members of the low-density lipoprotein receptor (LDLR) family. Localized in the endoplasmic reticulum and Golgi (Bu and Schwartz, Trends Cell. Biol. 8 (7):272-6, 1998), RAP acts as a chaperone for these family members. For example, RAP binds tightly to LRP in these compartments preventing premature association of the receptor with co-expressed ligands (Herz and Willnow, Atherosclerosis 118 Suppl:S37-41, 1995).
Full length human RAP including its signal sequence is 357 amino acids. Mature RAP contains 323 amino acids, of which the last four amino acids (HNEL (SEQ ID NO: 108)) constitute an endoplasmic reticulum retention signal. RAP has been reported to be composed of three weakly homologous domains (d1, d2 and d3). All three domains are capable of binding LRP, although d3 binds with the highest affinity. Different publications have reported slightly different assignment of the boundaries of the three domains of RAP (residues 1-100, 101-200 and 201-323, or 18-112, 113-218 and 219-323), and the domains appear to have slightly different binding characteristics. For example, d1 has been reported to inhibit binding of alpha-2-macroglobulin to LRP, while d3 has been reported to promote proper folding of LRP. (See Obermoeller et al., J. Biol. Chem. 272(16):10761-100768 (1997).) An alternative four-domain assignment (residues 1-92, 93-163, 164-216 and 217-323) has been reported by Medved et al., J. Biol. Chem., 274(2):717-727 (1999).
Truncated and/or mutated versions of RAP have been studied. Melman et al., J. Biol. Chem., 276 (31):29338-29346 (2001) reported the generation of a number of GST/RAP fragment fusions including residues 221-323, 221-275, 276-323, 221-290, 221-300 and 221-310 of mature RAP, which all exhibited no or low affinity binding to LRP. From this data, Melman et al. concluded that residues 201-210 were required for high affinity LRP binding. Melman et al. also generated RAP mutants containing mutations within positions 203-206 (site A), 282-289 (site B) and 314-319 (site C); mutations within site A alone or site B alone produced a small reduction in LRP binding activity, while mutations within both sites A and B significantly decreased LRP binding activity. Medved et al., supra, generated two GST/RAP C-terminal fragments fusions, one consisting of residues 216-323 of mature RAP and another consisting of residues 206-323. Rall et al., J. Biol. Chem., 273 (37):24152-24157 (1998) reported proteolytic cleavage of mature RAP into a variety of fragments and identified residues 223-323 as a highly protease resistant region. Obermoeller et al., supra, also generated a RAP fragment consisting of residues 191-323 of mature RAP and studied binding interactions with various domains of LRP. McCormick et al., Biochemistry, 44:5794-803 (2005) constructed a GSP/RAP fragment fusion containing residues 221-323 of RAP and evaluated its binding to endoplasmic reticulum chaperone ERp57. Andersen et al., Biochemistry 42, 9355-64 (2003) tested RAP domain fragments for binding to apoE receptor 2 and reported that only the third domain (residues 216-323) bound. Andersen et al., J Biol Chem 275, 21017-24 (2000) tested RAP d3 (residues 216-323) for binding to various fragments of LRP. Andersen et al., Biochemistry 40, 15408-17 (2001) also tested a variant of RAPd3, C-terminally truncated to residues 219-309, for ability to bind to an LRP fragment fused to ubiquitin (U-CR56), and observed a significant decrease in affinity for U-CR56. Lee et al. Mol Cell 22, 423-30 (2006) constructed mutants of full length RAP in which each conserved histidine was mutated to alanine, all histidines in domain 1 were mutated to alanine, all histidines in domain 2 were mutated to alanine, and all histidines in domain 3 were mutated to alanine. Migliorini et al., J Biol Chem 278, 17986-92 (2003) constructed a library containing clones with random mutations within residues 206-323 of RAP and reported that mutation of the lysine at position 256 or the lysine at position 270 of RAP abolished binding of RAP to LRP.
The lipoprotein receptor-related protein (LRP) receptor family comprises a group of cell-surface, transmembrane proteins that mediate a wide variety of physiological phenomena. All of the receptors are homologous to LDLR and share a similar domain organization, which includes groups of LDL receptor class A domains, or complement-type repeats (CR), that are part of a large family of conserved protein sequences. Structural data on members of this family suggests that CR sequences adopt a characteristic fold, the LDL receptor-like module (Structural Classification of Proteins, SCOP, terminology). CR sequences are found in a variety of different types of proteins including the LDLR family and the type II transmembrane serine protease (matriptase) family.
Mechanisms of action of LRP family members include both endocytosis of bound ligands and signal transduction from the extracellular space (1). LRP participate in various cellular functions, including but not limited to the metabolism of lipoproteins (2, 3), control of matrix metalloproteases and coagulation factors (4-6), specification of cell fate (3, 7), guidance of neural cell migration (7, 8), induction of proliferation in tumor cells (9, 10), binding of rhinovirus (11, 12), signalling by neurotransmitters (13, 14), acquisition of antigens by antigen presenting cells (15), trancytosis of ligands across the blood-brain barrier (16-19), recovery of proteins from glomerular filtrate (20), transport of endocrine hormones (21), efflux of amyloid β peptide from the brain (22), activation of bone deposition (23) and regulation of endothelial cell proliferation (24). The capacity of the LDLR to serve in so many roles derives in part from the diverse set of ligands to which these receptors are able to bind. Another feature of this receptor family is the diverse, and often unique, tissue distribution patterns of each LDLR.
The type II transmembrane serine protease family includes corin and the matriptases ST14, matriptase-2 and matriptase-3. Matriptase (MT-SP1, ST14, TADG-15) is overexpressed in a variety of epithelial tumors (carcinomas) (25-33). Following transactivation facilitated by hepatocyte activator inhibitor-1 (HAI-1), matriptase promotes tumor growth and metastasis by degrading extracellular matrix components directly or by activating other proteases, such as urokinase plasminogen activator (uPA), resulting in matrix-degradative events (26, 34, 35). In addition to the LDLR and matriptase families, a variety of other proteins have CR domains. One such protein, the FDC-8D6 antigen (CD320) has a pair of such domains and plays an important role in B-cell differentiation in lymphatic follicles (36, 37).
The important roles that CR-containing proteins play in pathophysiological processes, along with the unique tissue-distribution profiles of some members of these families, make these proteins useful drug targets. Protein-selective drugs could directly impact the function of a targeted protein, diminishing the supporting effects that the protein has on a particular disease state. Alternatively, the drug could take advantage of the tissue distribution of the targeted protein to efficiently deliver other therapeutic molecules to a particular tissue affected by a disease. Despite considerable evidence of the importance of CR-containing proteins in mammalian physiology and pathophysiology, there are few examples of drugs that act selectively on particular members of the LDLR or CR-containing protein families. The ability to create molecules that bind specific members of these families would provide a means of developing such drugs.
For example, WO 2006/138343 (Zankel et al.) reports data showing that a fusion of GDNF to RAP (which crosses the blood brain barrier) produced a conjugate that retained the disulfide-linked homodimeric conformation of GDNF. The RAP-GDNF conjugate bound and activated the receptor for GDNF (GFRα-1) with the same affinity (Kd) as GDNF, and retained biological activity in vitro as evidenced by RAP-GDNF induced neurite outgrowth in PC12 cells in culture.
Given the widespread participation of CR-containing proteins throughout mammalian physiology, there exists a need for RAP fragments and variants that retain the original binding characteristics of RAP, or that have improved binding selectivity for specific CR-containing proteins, and also exhibit other desirable properties such as improved stability or ease of production and manufacturing.